Synergistic clofazimine/metronidazole combination for treating clostridium difficile

ABSTRACT

Methods for treating a subject infected with  Clostridium difficile  comprises administering to the subject a synergistic combination of clofazimine and metronidazole. Pharmaceutical compositions comprising clofazimine and metronidazole are provided for treating  Clostridium difficile  infection and diseases or symptoms associated therewith.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a divisional application of U.S. application Ser.No. 14/947,212, filed Nov. 20, 2015, which claims priority benefit ofU.S. Provisional Applications No. 62/082,925, filed Nov. 21, 2014, andNo. 62/133,058, filed Mar. 13, 2015, which applications are incorporatedherein by reference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention relates to methods for preventing or treatingClostridium difficile associated disease (CDAD), and in particularmethods and compositions for treating CDAD by administering atherapeutically effective amount of clofazimine or analogues thereof,either alone or in combination with one or more additional therapeuticagents.

BACKGROUND OF THE INVENTION

Clostridium difficile (CD) is a Gram positive, spore-forming, anaerobicbacillus recognized to cause diarrhea, pseudomembraneous colitis, toxicmegacolon, other gastrointestinal conditions, and even death. Hedge etal. (2008) New advances in the treatment of Clostridium difficileinfection (CDI), Therapeutics and Clinical Risk Management 4(5):949-64;Tsutsumi et al. (2014) Progress in the Discovery of Treatments for C.difficile Infection: A Clinical & Medicinal Chemistry Review, CurrentTopics Med. Chem. 14:152-75. In its spore form, the bacterium is able tosurvive harsh environments and common sterilization techniques. Sporesof CD are resistant to high temperatures, ultraviolet light, harshchemicals, and many antibiotics, and remain viable for months or longer(Leffler & Lamont (2009) Treatment of Clostridium difficile-AssociatedDisease, Gastroenterol. 136:1899-1912).

Acquisition of CD occurs by ingestion of the acid-resistant spores,which pass through the stomach and germinate and inhabit the colon. Inorder for CD to over-populate the colon, there is typically a disruptionof the normal bacterial flora which otherwise provides colonizationresistance to CD and other opportunistic pathogens. As such, theprotection of normal gastrointestinal flora is an important defense toCD infection (CDI). Unfortunately, exposure to common antibioticregimens (e.g., cephalosporins, penicillins and fluoroquinolones) in thecourse of medical treatment of infectious and other diseases (e.g., suchas bowel surgery and/or cancer chemotherapy) disrupts the natural gutflora, allowing CD resistant to such antibiotics to colonize andoverpopulate in the gut of some patients, particularly elderly patients.

Once colonized, CD reproduces and releases enterotoxin (toxin A or TcdA)and cytotoxin (toxin B or TcdB) in the colon. Tsutsumi, 2014; Heinlen &Ballard (2010) Clostridium difficile Infection, Am. J. Med. Sci.340(3):247-52; Hedge et al., 2008. Both toxins A and B act asglucosyltransferases, inactivating small cellular GTPases (Rho, Rac &Cdc42), and triggering the attraction and adhesion of neutrophilsresulting in inflammation of the mucosal lining, cellular necrosis, andincreased peristalsis and capillary permeability, thereby degrading thecolonic epithelial cells and leading to the clinical symptoms associatedwith CDI. Although both toxins are cytotoxic and stimulate apoptosis,toxin B is more potent while toxin A stimulates epithelial cellpermeability and inflammatory response (effecting cytokine, chemokineand reactive oxygen intermediate production; neutrophil infiltration;mast cell accumulation; and substance P production, stimulatingsubmucosal sensory neurons).

The mechanisms of action of CD toxins TcdA and TcdB are illustrated inFIG. 1, plates A and B. See Voth & Ballard (2005) Clostridium difficileToxins: Mechanisms of Action and Role in Disease, Clin. Microbiol. Rev.18(2):247-63. Clinical symptoms vary from asymptomatic colonization ormild diarrhea to life threatening illness, including severeinflammation, lesions and/or tissue necrosis of the gut mucosa.Typically, CDI-associated disease begins as watery diarrhea andprogresses to pseudomembranous colitis. Distension of the colon mayresult in toxic megacolon. In addition, even cases of relatively mildCDI may rapidly progress to fulminant CDI, with such patients sufferingsystemic toxicity (e.g., leukocytosis, hypotension, renal failure,respiratory distress, or even death).

CD is a leading cause of hospital-acquired diarrhea, infections andother disease in Europe and North America. Heinlen & Ballard, 2010.Because of their increasing resistance to many common antibiotics, CDspores can remain in the gastrointestinal tract and potentiallycontribute to recurrent disease following conventional treatmentregimens. As such, CD infections (CDIs) are increasing worldwide andhave become more severe and refractory to treatment in the past decade.CDIs are one of the most common nosocomial infections in the UnitedStates, and presently, hospital-acquired CDIs exceed that ofmethicillin-resistant Staphylococcus aureus (MRSA) infections in someregions.

Increased incidence of hospital-associated CDI may be due to theutilization of a broad spectrum of antimicrobial agents to treat variousother conditions. As such, the emergence of hypervirulent epidemicstrains of CD (e.g., the fluoroquinolone resistant hypervirulent CDstrain designated as NAP1/BI/027 in North America and Europe) produceincreased amounts of toxins A and B, and thus exhibit increased rates ofrecurrence, morbidity and/or mortality (as compared to previouslyidentified strains of CD), particularly in vulnerable elderly patients.For example, NAP1/BI/027 produces 16 times more toxin A and 23 timesmore toxin B compared to many conventional CD strains, and also producesan additional toxin known as binary toxin (see Hedge et al., 2008),which causes a 3-fold higher mortality rate as compared to patientsinfected with less virulent strains. Leffler & Lamont, 2009.

Effective treatment of CDI has proven to be quite challenging due to theneed for selective eradication of CD without affecting the normal gutflora. After each episode or infection, the risk of recurrence of CDIgenerally increases. About 15%-30% of CDI patients get another infectionafter their first CDI, and about 40%-60% get a further CDI after theirsecond episode of the infection. Tsutsumi et al., 2014. As such, forpatients who experience two or more recurrences of symptomatic CDIs, achange in strategy is warranted. Leffler & Lamont, 2009.

Current therapies for CDI typically provide for the administration ofantimicrobial agents including metronidazole (MET), vancomycin (VAN) orfidaxomicin (FDX). MET is the conventionally accepted agent for treatingthe first episode of CDI due to its relatively low cost, as well asconcerns of emergence of VAN resistant enterococci (VRE) by extensiveVAN treatment. See Leffler & Lamont, 2009. However, only VAN and FDXhave been approved by the U.S. Food and Drug Administration (FDA) forthe treatment of recurrent CDI. Moreover, use of VAN and FDX isrelatively limited, particularly for treating the first episode of CDI,due to their relatively high cost (e.g., current cost is about $2000 pertreatment).

Failure to respond to MET has become more common recently, and thereforeVAN has become more commonly used as a first episode treatment,particularly in patients with more severe CDI. However, reportsdescribing MET failures and questions of the equivalence of MET and VANfor CDI have been raised. Hedge et al., 2008.

Recurrence or failure rate associated with conventional MET, VAN and FDXtreatments, as well as other conventional therapies, is relatively high.For example, between about 15% and about 30% of patients have a relapseof symptoms after successful initial treatment with such conventionaltreatments, usually within the first few weeks after treatment isdiscontinued Leffler & Lamont, 2009. Such relapse is sometimes due tothe persistence of the same CD strain being treated, or alternatively oradditionally due to reinfection with a new CD strain. With the emergenceof hypervirulent epidemic strains of CD, recurrence rates of CDI havejumped to nearly 50% in patients treated with MET and VAN. The spectrumof activity for FDX is narrower compared to that of MET or VAN. However,efficacy of FDX is similar to that of VAN. When used to treat previousstrains of CD, FDX exhibits a somewhat lower recurrence or failure rateas compared to MET and VAN. However, FDX exhibits no improvement inrecurrence rates in patients infected with hypervirulent strains of CD(Louie et al. (2011) Fidaxomicin versus vancomycin for Clostridiumdifficile infection, N. Engl. J. Med. 364(5):422-31), and thus alsoexhibits recurrence rates of nearly 50%. As such, conventional FDXtherapies have not proven to be effective for patients that havesuffered multiple CDI relapses.

Thus, due to the increasing emergence of resistant and hyper-virulent CDstrains and the high rate of recurrence of CDI after treatment with suchconventional agents, there is an urgent need to develop new and bettertherapies. Alternative non-antibiotic based treatments for treating CDIhave emerged, including fecal transplants and probiotics, which seek torestore or repopulate the normal gut flora. Other treatment methods,such as immunotherapy-based treatments and vaccines, attempt toneutralize the CD toxins or enhance the patient's immune response.However, such non-antibiotic based treatments have exhibited mixedresults with less efficacy as compared to conventional microbial agents.As such, conventional treatments using MET, VAN and FDX remainprevalent.

Thus, new therapeutics and treatment methods are needed to improveefficacy and reduce failure and/or recurrence rates of CDIs. The idealtherapeutic agent would specifically eliminate or substantially reduce aCD population with minimal disturbance to normal gut flora. However, dueto the exorbitant costs involved in the discovery and development of newagents, many pharmaceutical companies are reluctant to invest in newdrug discoveries. Moreover, the return on investment for drug companieson short duration therapeutics like antimicrobial agents is not alluringcompared to long duration or life-long therapies. Consequently, thediscovery of new uses for previously known drugs (repurposing) is a verycost effective option. Indeed, the National Institutes of Health has anongoing initiative in collaboration with pharmaceutical companies fordiscovering new therapeutic uses for existing molecules.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods fortreating CDI and/or CDAD. According to an embodiment of the presentinvention, a method of preventing or treating a subject infected with CDcomprises administering to the subject a therapeutically effectiveamount of clofazimine (CFM) or a CFM derivative or analogue(s) includingbut not limited to B746, B4157, and B4129, either alone or incombination with one or more additional therapeutic agents. In someimplementations, the compound is administered orally. In otherimplementations, the compound is administered parenterally. In someembodiments, for determining in vitro activity, the compound isadministered in a concentration ranging from about 0.020 μg/ml to about1.0 μg/ml, more preferably from about 0.031 μg/ml to about 0.25 μg/ml.

In some embodiments, the compound is administered in connection with apharmaceutically acceptable carrier and/or excipient. In someimplementations, a pharmaceutical composition suitable foradministration in accordance with disclosed embodiments consists solelyof a single active agent selected from the group consisting of CFM, anda CFM analogue including B746, B4157 and B4129. In otherimplementations, the pharmaceutical composition comprises CFM and/or aCFM analogue(s) including B746, B4157 and B4129, and/or one or moreadditional therapeutic agents (e.g., an antimicrobial agent, includingbut not limited to MET, VAN and/or FDX).

In one embodiment, a method of treating a subject infected withClostridium difficile comprises administering to the subject atherapeutically effective amount of clofazimine or a clofazimineanalogue. In some implementations, the clofazimine analogue is selectedfrom the group consisting of B4154 analogue, B4129 analogue, B4087analogue, B4165 analogue, B826 analogue, B3640 analogue, B4100 analogue,B4101 analogue, B4021 analogue, B4157 analogue, B746 analogue, and B3987analogue. In preferred embodiments, the clofazimine analogue is B4129analogue, B4157 analogue, or B746 analogue.

In some embodiments, the disclosed method provides for administering thecompound orally. The compound may be administered in a concentrationfrom about 0.020 μg/ml to about 1.0 μg/ml for in vitro conditions, morepreferably in a concentration from about 0.031 μg/ml to about 0.25 μg/mlfor in vitro conditions.

In some embodiments, the compound is administered in combination with atleast one additional therapeutic agent. In some implementations, theadditional therapeutic agent is an antimicrobial agent, including butnot limited to vancomycin, metronidazole, or fidaxomicin.

In another embodiment, a method of treating a subject infected with apopulation of Clostridium difficile comprises administering to thesubject a pharmaceutical composition comprising: (A) an effective amountof a first antimicrobial agent comprising clofazimine or a clofazimineanalogue; (B) an effective amount of a second antimicrobial agent; and(C) a pharmaceutically acceptable carrier or excipient. The effectiveamounts of the first and second antimicrobial agents cause thepharmaceutical composition to mediate a synergistically increasedreduction in the population of Clostridium difficile relative to thereductions in the population mediated by: (1) a pharmaceuticalcomposition comprising said effective amount of said first antimicrobialagent but lacking said effective amount of said second antimicrobialagent; and (2) a pharmaceutical composition comprising said effectiveamount of said second antimicrobial agent but lacking said effectiveamount of said first antimicrobial agent.

In some embodiments, the pharmaceutical composition comprises aclofazimine analogue selected from the group consisting of B4154analogue, B4129 analogue, B4087 analogue, B4165 analogue, B826 analogue,B3640 analogue, B4100 analogue, B4101 analogue, B4021 analogue, B4157analogue, B746 analogue, and B3987 analogue. Preferably, the clofazimineanalogue is B4129 analogue, B4157 analogue, or B746 analogue.

In some embodiments, the first antimicrobial agent is administered in aconcentration from about 0.020 μg/ml to about 1.0 μg/ml for in vitroconditions, more preferably in a concentration from about 0.031 μg/ml toabout 0.25 μg/ml for in vitro conditions.

In some embodiments, the second antimicrobial agent is selected from thegroup consisting of vancomycin, metronidazole, and fidaxomicin.

The present invention also relates to a pharmaceutical composition forkilling or reducing a population of Clostridium difficile comprising atherapeutically effective amount of clofazimine or a clofazimineanalogue, and a pharmaceutically acceptable carrier or excipient. Theclofazimine analogue is preferably selected from the group consisting ofB4154 analogue, B4129 analogue, B4087 analogue, B4165 analogue, B826analogue, B3640 analogue, B4100 analogue, B4101 analogue, B4021analogue, B4157 analogue, B746 analogue, and B3987 analogue. Morepreferably, the clofazimine analogue is B4129 analogue, B4157 analogue,or B746 analogue.

In some embodiments, the pharmaceutical composition also comprises atleast one additional therapeutic agent. In some implementations, theadditional therapeutic agent is an antimicrobial, an antibiotic, or alytic enzyme. In some implementations, the additional therapeutic agentis an antimicrobial agent selected from the group consisting ofvancomycin, metronidazole, and fidaxomicin.

The present invention also provides for use of the disclosed compoundscomprising clofazimine and/or a clofazimine analogue(s), and/or use ofthe disclosed pharmaceutical composition(s) in the treatment orprevention of a disease or condition associated with Clostridiumdifficile infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates schematically mechanisms of action of CD toxins. Anoverview of intracellular modifications by the CD toxins (TcdA and TcdB)is illustrated in plate A. TcdA and TcdB toxins enter the cell throughreceptor-mediated endocytosis and require an acidified endosome fortranslocation. The requirement for low pH is believed to be due toimportant structural changes which occur in the toxins, leading toexposure of hydrophobic domains prior to insertion into the targetmembrane. Both TcdA and TcdB act intracellularly asglycosyltransferases. Each toxin modifies and inactivates Rho, Rac, andCdc42 via transfer of a sugar moiety, with UDP-glucose as aco-substrate. The effects of these modifications include actincondensation, transcriptional activation, and apoptosis. Downstreameffects of TcdA and TcdB in intestinal cells during disease is shown inplate B. Exposure of intestinal epithelial cells to TcdA leads toneutrophil infiltration, substance P production, chemokine production,reactive oxygen intermediate production, disruption of tight junctions,and apoptosis. TcdB activity leads to disruption of tight junctions andapoptosis. A combination of one or more of these activities leads tofluid accumulation in the host and inflammatory responses.

FIG. 2 illustrates the chemical structures of: FIG. 2(a) Clofazimine;FIG. 2(b) B746 analogue; FIG. 2(c) B4157 analogue; and d) B4129analogue.

FIG. 3 illustrates the chemical structure of azaquinone.

FIG. 4 shows minimum inhibitory concentration (MIC) determinations bybroth microdilution method against CD strain VPI 10463. Panel (A)depicts MIC results against CD strain VPI 10463 in brain heart infusionbroth. Panel (B) depicts MIC results against CD strain VPI 10463 inbrucella broth. The different drugs tested are identified along the leftof panels (A) and (B) and presented in rows A-F (Row A, clofazimine(CFM); Row B, clofazimine analogue B746; Row C, clofazimine analogueB4157; Row D, clofazimine analogue B4129; Row E, vancomycin (VAN); RowF, metronidazole (MET); Rows G and H were empty. Differentconcentrations of the drugs are presented along the top of panels (A)and (B) and in Columns 1-11, with 32 μg/ml concentration in Column 1,with decreasing concentration in columns from left to right, to 0.031μg/ml concentration in Column 11. A drug-free control is presented inColumn 12. The minimal inhibitory concentration (MIC) [lowestconcentration showing no sign of visible growth] of each drug isidentified along the right of panels (A) and (B). MIC (μg/ml) resultsagainst the strain in brain heart infusion broth are as follows: CFM,0.062; B746, 0.125; B4157, ≦0.031; B4129, ≦0.031; VAN, 0.25; and MET,0.062. MIC (μg/ml) results against the strain in brucella broth are asfollows: CFM, 0.125; B746, 0.125; B4157, ≦0.031; B4129, ≦0.031; VAN,0.25; and MET, 0.125.

FIG. 5 illustrates graphically MIC determinations presented in FIG. 4.

FIG. 6 illustrates graphically MIC (μg/ml) for 13 riminophenazinecompounds against five CD strains, including CFM, 12 CFM analogues(B4154; B4129; B4087; B4165; B826; B3640; B4100; B4101; B4021; B4157;B746; and B3987), VAN and MET. Five bars above each compound testedrepresent, from left to right: strain 1 (VPI10463), strain 2 (NR32886),strain 3 (NR32887), strain 4 (NR32882) and strain 5 (NR32889),respectively (for example, such as identified above analogue B3987).

FIG. 7 illustrates graphically portions of MIC data shown in FIG. 6,with MIC data greater than 8 μg/ml removed in order to show MIC data ≦2μg/ml in further detail.

FIG. 8 illustrates graphically MIC (μg/ml) data for CFM, CFM analogues(B746, B4157, and B4129), VAN and MET against CD strains in brain heartinfusion broth.

FIG. 9 is an image of sample vials of supernatants of CFM-DMSO, CFM-F68,CFM-PEG, and CFM CrEL formulations.

FIG. 10 is an image of the sample vials of the supernatants of FIG. 9incubated with fecal extracts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to pharmaceutical compositions andmethods for preventing or treating CDI and/or CDAD. According toembodiments of the present invention, pharmaceutical compositions fortreating CDI and/or CDAD comprise clofazimine (CFM) and/or a CFManalogue(s), either as the primary or sole active compound or incombination with one or more additional therapeutic agents. According toother embodiments, pharmaceutical compositions for treating CDI and/orCDAD additionally or alternatively comprise azaquinone (AZQ) (also knownas Gangamicin, NSC 186017, and BRN 0407295).

CFM (see FIG. 2(a)),3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-iso-propyliminophenazine,is a fat-soluble riminophenazine dye, which was discovered and developedas a treatment for tuberculosis (TB) in the 1950s. However, because ofits low activity against TB in guinea pig and simian models, theinterest in the drug as an effective treatment for TB quicklydiminished, although CFM continues to be used for treating multidrugresistant TB. See Cholo et al. (2011) Clofazimine: current status andfuture prospects, J. Antimicrob. Chemother. 67:290-98.

In addition, in its micronized form, CFM was discovered to be effectiveagainst leprosy. Since then, it has continued to be used for treatingmultibacillary leprosy in a multidrug regimen consisting of dapsone,rifampin and CFM (Reddy et al. (1999) Antimycobacterial activities ofriminophenazines, J. Antimicrob. Chemother. 43:615-23), and has beenparticularly effective for treating immune mediated conditions (e.g.,erythema nodosum leprosum (ENL), Mycobacterium avium complex (MAC)disease, rhinoscleroma, pyoderma gangrenosum, necrobiosis lipoidica,severe acne, pustular psoriasis, and lupus erythematosus). CFM has alsobeen used effectively in the treatment of Crohn's disease. Feller et al.(2010) Long-Term Antibiotic Treatment for Crohn's Disease: SystematicReview and Meta-Analysis of Placebo-Controlled Trials, Clin. Infect.Dis. 50:473-80). However, anaerobic bacteria (such as CD) have not beenimplicated in the pathogenesis of Crohn's disease. Id.

CFM is believed to kill bacteria by disruption or destabilization ofbacteria membrane function. In particular, CFM selectively accumulatesin bacterial membranes and stimulates reactive oxygen species, blocks K⁺channels and/or enhances bacterial phospholipase A₂ activity and therelease of lysophospholipids. CFM is virtually insoluble in water(solubility 0.225 mg/L). It has an orally absorbed bioavailability ofbetween about 45% and 65%. CFM has a half-life of about 10 days after asingle dose of 100 mg, and about 70 days following long-term high dosageof 300 mg. For example, recommended dosage of CFM for treating leprosyis between 100-300 mg daily for 2-3 years in combination with otherantileprosy drugs (dapsone and rifampin). CFM exhibits low plasmaconcentration, but relatively high tissue concentration. CFM is highlylipophilic and deposits primarily in fatty tissues and thereticuloendothelial system throughout the body. It is metabolized in theliver forming three metabolites, and excreted mainly in the feces,although it is detectable in all body secretions.

Various adverse effects associated with treatment of leprosy and TBusing CFM have been reported (Reddy et al., 1999) including: skinpigmentation (approximately 75-100% of patients); icothyosis and drynessof skin (approximately 8-28% of patients), rash and pruritus(approximately 1-5% of patients); gastrointestinal conditions includingabdominal and epigastric pain, nausea, vomiting and/or diarrhea(approximately 40-50% of patients); ocular conditions includingconjunctival pigmentation, itching and dryness (approximately 1% ofpatients). Such adverse effects are typically reported, if at all, afterseveral weeks of treatment, and are generally reversible and dissipateupon cessation of treatment.

In accordance with disclosed embodiments, the duration of treatment forCD is significantly shorter (e.g., 7-10 days) as compared to theduration of treatment for leprosy, TB or other chronic diseases whereinCFM is used. The possibility of such reported adverse effects associatedwith CFM are therefore minimized or eliminated when used in accordancewith disclosed embodiments of treatment.

Compositions and methods utilizing or including CFM and/or CFM analoguesare effective in killing or reducing CD populations and in particularfor treating CDI and/or CDAD. In some implementations, pharmaceuticalcompositions include CFM and/or a CFM analogue(s) as the primary or soleactive ingredient(s) or compound. In other implementations,pharmaceutical compositions include CFM and/or a CFM analogue(s) incombination with one or more additional therapeutic agents.

As demonstrated, the disclosed compounds exhibit excellent in vitroactivity against CD. In vitro activity of antimicrobial agents may beexpressed in terms of a minimal inhibitory concentration (MIC), which isconsidered to be the lowest concentration effective in preventingfurther bacterial growth, or killing or substantially reducing abacterial population. MIC₅₀ represents the MIC of the active compound(s)effective against 50% of tested isolates; MIC₉₀ represents the MIC ofthe active compound(s) effective against 90% of tested isolates.

In accordance with disclosed embodiments, AZQ also exhibited excellentin vitro activity against CD, and is thus demonstrated herein to be aneffective bactericidal specific for CD treatment and/or for use inpharmaceutical compositions for treating CDI and/or CDAD. AZQ (FIG. 3),6-(cyclooctylamino)-5,8-quinolinequinone (CQQ) is an analogue of theubiquinone (co-enzyme Q) and acts as an inhibitor of the cell-wallsynthetic process. AZQ is known to be active against M. tuberculosis andM. avium. Hart et al. (1996) Tuberculosis into the next century, J. Med.Microbiol. 44:1-34; U.S. Pat. No. 4,963,565. Chemical and physicalproperties of AZQ are presented in Table 1 below:

TABLE 1 AZQ Properties Molecular Weight 284.3529 g/mol Molecular FormulaC₁₇H₂₀N₂O₂ XLogP3 3.8 Hydrogen Bond Donor Count 1 Hydrogen Bond AcceptorCount 4 Rotatable Bond Count 2 Tautomer Count 3 Exact Mass 284.152478g/mol Monoisotopic Mass 284.152478 g/mol Topological Polar Surface Area59.1 A{circumflex over ( )}2 Heavy Atom Count 21 Formal Charge 0Complexity 436 Isotope Atom Count 0 Defined Atom Stereocenter Count 0Undefined Atom Stereocenter Count 0 Defined Bond Stereocenter Count 0Undefined Bond Stereocenter Count 0 Covalently-Bonded Unit Count 1Feature 3D Acceptor Count 3 Feature 3D Donor Count 1 Feature 3D CationCount 1 Feature 3D Ring Count 2 Effective Rotor Count 3.6 ConformerSampling RMSD 0.6 CID Conformer Count 53

In accordance with disclosed embodiments, methods of treating a subject(e.g., a mammal such as a human) infected with CD provide foradministering to such subject a therapeutically effective amount of CFM,a CFM analogue (e.g., B746, B4157, B4129), and/or AZQ. In someimplementations, the subject is administered a pharmaceuticalcomposition comprising CFM, a CFM analogue(s), and/or AZQ, optionallyone or more additional therapeutic agents, and a pharmaceuticallyacceptable carrier and/or excipient. Such composition(s) may beadministered to the subject using an administration technique known tothose of skill in the art (e.g., orally or intravenously), as discussedin further detail below.

The present invention provides for pharmaceutical compositionscontaining therapeutically effective amounts of CFM and/or a CFManalogue(s). The pharmaceutical compositions of the present inventionmay include a secondary therapeutic agent in addition to therapeuticallyeffective amounts of CFM and/or a CFM analogue(s), such as for examplean additional antimicrobial, antibiotic, and/or lytic enzyme. Thepharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in REMINGTON: SCI. & PRACTICE OF PHARM., 21th Edition,Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2005.

The pharmaceutically acceptable carriers or diluents, as well as anyother known adjuvants and excipients, e.g., such as preservatives,wetting agents, emulsifying agents, dispersing agents, preservatives orbuffers, may enhance shelf life or effectiveness of the pharmaceuticalcomposition. Such carriers, diluents and/or other adjuvants should besuitable for the chosen compound(s) of the present invention and thechosen mode of administration. Suitability for carriers and othercomponents of pharmaceutical compositions is determined based on thelack of significant negative impact on the desired biological propertiesof the chosen compound or pharmaceutical composition of the presentinvention (e.g., less than a substantial impact (10% or less relativeinhibition, 5% or less relative inhibition, etc.)).

The pharmaceutical compositions of the present invention may thusinclude diluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in thecomposition. The diluent is selected to not affect the biologicalactivity of the combination. Examples of such diluents are distilledwater, physiological phosphate-buffered saline, Ringer's solutions,dextrose solution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, ornon-toxic, nontherapeutic, non-immunogenic stabilizers and the like. Thecompositions may also include large, slowly metabolized macromolecules,such as proteins, polysaccharides like chitosan, polylactic acids,polyglycolic acids and copolymers (e.g., latex functionalized sepharose,agarose, cellulose, and the like), polymeric amino acids, amino acidcopolymers, and lipid aggregates (e.g., oil droplets or liposomes).

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial agents, isotonicityagents, antioxidants and absorption delaying agents, and the like thatare physiologically compatible with a compound of the present invention.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude saline, phosphate buffered saline, ethanol, dextrose, polyols(such as glycerol, propylene glycol, polyethylene glycol, and the like),and suitable mixtures thereof, vegetable oils, such as olive oil, cornoil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulosecolloidal solutions, tragacanth gum and injectable organic esters, suchas ethyl oleate, and/or various buffers. Other carriers are well knownin the pharmaceutical arts and may alternatively or additionally beincluded.

The compounds of the present invention may be prepared with carriersthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Such carriers may includegelatin, glyceryl monostearate, glyceryl distearate, biodegradable orbiocompatible polymers such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid aloneor with a wax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., SUSTAINED & CONTR. RELEASE DRUG DELIV. SYS., J. R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Pharmaceuticalcompositions of the present invention may also comprise isotonicityagents, such as sugars, polyalcohols, such as mannitol, sorbitol,glycerol or sodium chloride.

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution and efficacy in vivo.Pharmaceutically acceptable carriers for parenteral administration mayinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound(s), use thereof in thepharmaceutical compositions of the present invention is contemplated.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionsmay be formulated as a solution, microemulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe a aqueous or nonaqueous solvent or dispersion medium containing forinstance ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In some cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition.

Prolonged absorption of the compositions may be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. Sterile solutions may beprepared by incorporating the active compound in the required amount inan appropriate solvent with one or a combination of ingredients, e.g.,as enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients, e.g., from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The pharmaceutical compositions may be prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, examples of methods of preparation are vacuum drying andfreeze-drying (lyophilization) that yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Methods of treating CDI and/or CDAD in a patient in accordance with thepresent invention comprise administering to the patient atherapeutically effective amount of CFM and/or a CFM analogue(s), eitheralone or in combination with one or more additional therapeutic agentsas noted above. The term “treat” or “treating” a disease, including aninfectious disease or infection, refers to killing or reducing thegrowth of the bacteria (e.g., CD) causing such disease or infection,and/or reducing, ameliorating or eliminating symptoms associated withsuch disease or infection. A “therapeutically effective amount” refersto an amount of active compounds (e.g., CFM, CFM analogue(s) and/or oneor more additional therapeutic agents) sufficient to elicit a desiredbiological response in a subject, and in particular an amount sufficientto kill, reduce or stabilize a bacterial population causing an infectionor related disease and/or sufficient to reduce symptoms associated withsuch infection or disease. Preferably, a therapeutically effectiveamount of the active compounds of the present invention is effective inreducing growth of the bacterial population by at least about 50%, morepreferably by at least about 75%, most preferably by at least about 90%or more.

In some embodiments, a method of treating or preventing CDI and/or CDADprovides for administering to a subject a pharmaceutical compositioncomprising CFM, a CFM analogue(s) and/or AZQ, along with one or moreadditional therapeutic agents, such as an additional antimicrobial agent(e.g., FDX, VAN or MET), an antibacterial, an antibody, a cell bindingmotif, or an antibiotic.

The efficacy of the disclosed pharmaceutical compositions and methodsherein may be enhanced if more of the active compounds remain in the gut(as opposed to absorbing into the bloodstream). In some embodiments, thedisclosed compounds and pharmaceutical compositions may be administeredto a subject on an empty stomach (e.g., ½ hour, more preferably 1-2hours, before or after ingesting any food or drinks, excluding water).

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time, or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. Pharmaceutical compositions may be formulated in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the patients to be treated, with each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and dependent on thecharacteristics of the active compound and the particular therapeuticeffect to be achieved, as well as any limitations in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

A physician having ordinary skill in the art may readily determine andprescribe the therapeutically effective amount of the pharmaceuticalcomposition required for a particular patient. The actual dosage levelsof the active ingredient(s) in the pharmaceutical compositions of thepresent invention may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known to those in the medical arts.

In addition, the therapeutically effective amount is one in which anytoxic or detrimental effects of the pharmaceutical composition areoutweighed by the therapeutically beneficial effects. The physician maystart doses of pharmaceutical compositions comprising the activecompounds of the present invention at levels lower than that required inorder to achieve the desired therapeutic effect and gradually increasethe dosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the present invention will be that amountof the compound which is the lowest dose effective to produce thedesired therapeutic effect (e.g., killing or reducing a population of CDbacteria), and/or for treating or preventing infection, and/or forameliorating or alleviating symptoms associated with such bacteria in asubject). Such an effective dose will generally depend upon the factorsdescribed above.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound(s) as apharmaceutical composition as described above. The pharmaceuticalcompositions of the present invention may be administered by anysuitable route and mode, including: parenteral, topical, oral orintranasal for prophylactic and/or therapeutic treatment. In oneembodiment, a pharmaceutical composition of the present invention isadministered orally. In another embodiment, a pharmaceutical compositionof the present invention is administered parenterally. The phrases“parenteral administration” and “administered parenterally” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection (e.g., including epidermal,intravenous, intramuscular, intraperitoneal, subcutaneous, etc.).Additional suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the medical arts. Thepharmaceutical compositions and compounds in accordance with the presentinvention may be administered via capsules, tablets, lozenges, chewinggums, powders, sprays, liquids, ointments, etc.

While some embodiments are described with respect to use in humans, thepharmaceutical compositions and methods of the present invention arealso suitable for veterinary (non-human) applications. For example,studies have isolated CD from meat products intended for consumption byhumans or pets. See Songer et al. (2009) Clostridium difficile in retailmeat products, USA, 2007, Emerg. Infect. Dis. 15(5):819-21. Thepharmaceutical compositions and compounds of the present invention maybe utilized for treating bacterial infection or contamination inlivestock or other animals (e.g., by administration to such livestock oranimal orally, nasally, parenternally, onto the skin or coat, viaintramammary infusion, teat dip, etc. as described herein).

Additional characteristics and features of the present invention will befurther understood through reference to the following additionaldiscussion and examples, which are provided by way of furtherillustration and are not intended to be limiting of the presentinvention.

Minimal Inhibitory Concentration Screenings:

In initial screenings, minimum inhibitory concentration (MIC) of CFM andCFM analogues (the test compounds) were determined by brothmicrodilution method in two different media, brucella broth (BB) andbrain heart infusion broth (BHI), wherein serial dilutions of the testcompounds were prepared in 100 μl volumes of BB/BHI broth in 96-wellmicrotiter plates. BB and BHI are suitable for the cultivation of mostanaerobic bacteria and other fastidious microorganisms; however, BBincluding lysed blood cells provides data in test environment muchcloser to in vivo conditions as compared to BHI test data.

Referring to FIG. 4 and FIG. 5, MIC was determined for CFM, analoguesB746, B4157 and B4129, as well as VAN and MET by broth microdilutionmethod against CD strain VPI-10463. MIC results in BHI are shown in FIG.4, panel (A). MIC results in BB are shown in FIG. 4, panel (B).

The drug dilutions were kept in the Bactron EZ anaerobic chamber (ShelLab., Cornelius, Oreg.) for 4-6 hours to reduce. Overnight growth of CDstrains in BHI were adjusted to 0.1 OD at

600, diluted 1:20 in BB/BHI, and 100 μl volumes were dispensed to eachwell. Positive controls of VAN and MET were run in parallel with eachtest. The microtiter plates were placed in zip-lock bags and left in theanaerobic chamber at 37° C. for 48 hours before reading. The lowestconcentration at which each test compound showed no visible growth wasdetermined to be the MIC. (The MIC of the test compounds against aerobicbacteria was also determined by broth microdilution method inMuller-Hinton broth using the same protocol).

With continued reference to FIG. 4, panels (A) and (B), each rowcontains a tested drug: row A, clofazimine (CFM); row B, clofazimineanalogue B746; row C, analogue B4157; row D, analogue B4129; row E,vancomycin (VAN); row F, metronidazole (MET). Rows G and H were empty(not used). The tested drugs were diluted in culture medium from left toright; thus, each column of wells contained a different concentration ofthe drug starting from 32 μg/ml in column 1 (the left most column), 16μg/ml in column 2, and so forth, through 0.031 μg/ml in column 11.Column 12 (the right most column) was a drug-free control. Addition ofbacteria to each well of the plates and their incubation for 48 hourswas under anaerobic conditions. At the end of incubation, clear wellsindicated no visible bacteria growth, while white or gray deposits inthe wells indicated growth (see FIG. 4, panels (A) and (B)).

The lowest concentration of the drug showing no visible growth wasdetermined to be MIC. As shown in FIG. 4, panel (A), row A containingCFM, wells in columns 1 through 10 indicate no visible growth, while thewell in column 11 shows visible CD growth. Thus, the well in column 10containing 0.062 μg/ml had the lowest concentration of CFM showing novisible bacterial growth—therefore, MIC of CFM was considered to be0.062 μg/ml in BHI (panel (A)).

MIC of CFM against various tested CD strains ranged from ≦0.031 to 0.25μg/ml, with MIC₅₀ determined to be 0.062 μg/ml and MIC₉₀ determined tobe 0.25 μg/ml. CFM demonstrated relatively low activity against otherGram-positive bacteria tested as presented below (e.g., Enterococcusfaecalis, Enterococcus faecium, and Staphylococcus aureus), and was notactive against aerobic Gram-negative bacteria tested (e.g., Escherichiacoli). As such, CFM and particular analogues of CFM demonstratedeffective bactericidal activity against and specific for CD. CFM andAnalogue Activity Against CD Strains:

Thirteen riminophenazine compounds, including CFM and 12 CFM analogues(B4154, B4129, B4087, B4165, B826, B3640, B4100, B4101, B4021, B4157,B746, B3987), as well as VAN and MET, were screened to determineactivity against five CD strains. Twelve out of thirteenriminophenazines demonstrated good activity with MICs of ≦0.5 μg/ml, asshown in Table 2 and graphically in FIG. 6 and FIG. 7.

TABLE 2 MIC of CFM and CFM Analogues against CD strains MIC (μg/ml)against CD strain Analog: VPI10463 NR32886 NR32887 NR32882 NR32889 CFM0.125 0.125 0.125 0.125 0.062 B4154 0.5 0.125 0.125 0.25 0.25 B4129≦0.031 0.125 0.062 ≦0.031 0.062 B4087 0.062 0.125 0.062 0.062 0.062B4165 0.125 0.125 0.25 0.25 0.25 B826 0.5 0.25 0.25 0.25 0.25 B3640 0.50.125 0.25 0.25 0.25 B4100 0.25 0.125 0.25 0.25 0.25 B4101 0.062 0.0620.062 0.125 0.062 B4021 0.125 0.062 0.125 0.25 0.062 B4157 ≦0.031 0.062≦0.031 0.062 0.062 B746 0.125 0.062 0.125 0.125 0.125 B3987 16 8 16 16 8VAN 0.25 0.25 1 0.5 1 MET 0.125 0.25 8 2 8 BB = Brucella broth; BHI =brain heart infusion; CFM = clofazimine; VAN = vancomycin; MET =metronidazole; the rest are CFM analogs

Following the initial screen of the 13 riminophenazine compounds (Table2), four compounds (CFM, and CFM analogues B746, B4157, and B4129) wereselected for further testing against a panel of additional CD strains.MIC data for CFM and analogues B746, B4157 and B4129 against various CDstrains in BHI, as well as MICs for VAN and MET, were determined, aspresented in Table 3 and graphically in FIG. 8.

TABLE 3 MIC of CFM and CFM Analogues against CD strains in BHI MIC(μg/ml) Strain: CFM B746 B4157 B4129 VAN MET HM89 0.25 0.25 0.125 0.1250.5 0.5 HM745 0.25 0.25 ≦0.031 0.125 0.5 0.5 HM746 0.5 0.5 0.062 0.1250.5 1 NR13427 1 1 ≦0.031 0.125 2 0.5 NR13431 0.25 0.25 0.062 0.125 0.252 NR13434 0.25 0.125 ≦0.031 ≦0.031 0.25 0.25 NR13436 0.5 0.5 0.25 0.250.5 0.5 NR13438 0.25 0.25 0.125 0.125 1 0.5 NR32882 0.125 0.25 0.0620.062 0.5 1 NR32883 0.25 0.125 0.062 0.062 0.5 1 NR32884 0.062 0.1250.25 0.062 0.5 0.5 NR32885 0.25 0.125 0.062 ≦0.031 1 0.5 NR32888 0.062≦0.031 0.062 0.062 0.5 1 NR32892 0.25 0.062 ≦0.031 0.062 1 1 NR328950.125 0.125 0.062 ≦0.031 2 2 NR32903 0.125 0.125 0.062 0.062 1 1 NR329040.062 0.062 ≦0.031 ≦0.031 0.5 1 NR13553 ≦0.031 0.062 ≦0.031 ≦0.031 0.50.25 VPI 10463 0.125 0.125 ≦0.031 ≦0.031 0.5 0.062 BAA1805* 0.125 — — —1 1 C. perfringens 1 1 2 2 0.5 1 MH310 MIC determined by brothmicrodilution method; CFM = clofazimine; B746, B4157, B4129 are CFManalogues; VAN = vancomycin; MET = metronidazole. *Hyper-virulent strainBI/NAP1/027

In addition, MICs of CFM and analogues B746, B4157 and B4129, as well asMICs for VAN and MET, against the various CD strains in BB weredetermined, as presented in Table 4:

TABLE 4 MIC of CFM and CFM Analogues against CD strains in BB MIC(μg/ml) Strain CFM B746 B4157 B4129 VAN MET HM88 0.062 0.062 0.125 0.0620.5 1 HM89 0.062 0.125 0.062 ≦0.031 0.5 2 HM745 0.062 0.125 0.062 ≦0.0311 2 HM746 0.125 0.125 0.062 ≦0.031 1 2 HM747 0.062 0.125 ≦0.031 ≦0.0310.5 0.5 NR13427 0.125 0.125 0.062 0.062 2 8 NR13428 ≦0.031 0.125 ≦0.031≦0.031 1 0.5 NR13429 0.062 0.062 ≦0.031 ≦0.031 0.5 4 NR13430 0.062 0.0620.062 0.062 1 1 NR13431 0.125 0.125 0.062 ≦0.031 0.5 2 NR13432 0.0620.125 ≦0.031 ≦0.031 2 2 NR13433 0.062 0.062 ≦0.031 ≦0.031 0.5 0.125NR13434 ≦0.031 ≦0.031 ≦0.031 ≦0.031 0.5 0.5 NR13435 0.062 0.062 ≦0.031≦0.031 0.5 0.5 NR13436 0.125 0.125 0.125 ≦0.031 2 2 NR13437 0.125 0.0620.062 ≦0.031 0.5 1 NR13438 0.25 0.5 ≦0.031 ≦0.031 2 2 NR13553 0.1250.062 ≦0.031 ≦0.031 0.5 0.5 NR32882 0.25 0.25 0.25 0.25 0.5 0.5 NR32883≦0.031 0.062 ≦0.031 0.062 0.5 1 NR32884 0.25 0.125 0.125 0.125 1 0.5NR32885 0.125 0.062 ≦0.031 ≦0.031 2 1 NR32886 ≦0.031 0.062 ≦0.031 ≦0.0311 1 NR32887 ≦0.031 0.062 ≦0.031 ≦0.031 1 8 NR32888 0.25 0.5 ≦0.031 0.0620.5 1 NR32889 0.125 0.25 0.062 0.062 2 8 NR32890 0.062 ≦0.031 0.062≦0.031 1 1 NR32891 0.062 0.125 0.062 ≦0.031 0.5 1 NR32892 0.25 0.1250.125 0.062 0.5 2 NR32895 0.125 0.5 0.125 0.062 2 4 NR32896 0.062 0.125≦0.031 ≦0.031 2 1 NR32897 ≦0.031 0.062 ≦0.031 ≦0.031 0.25 2 NR329000.125 0.062 ≦0.031 ≦0.031 0.5 1 NR32903 0.125 0.125 ≦0.031 0.125 0.5 0.5NR32904 0.125 0.125 0.062 0.125 0.5 2 VPI 10463 0.125 0.125 ≦0.031≦0.031 1 0.125 BAA1805* 0.125 0.25 0.125 0.062 0.5 8 C. perfringens 0.50.5 0.5 0.25 0.25 2 MH310 MIC determined by broth microdilution method;CFM = clofazimine; B746, B4157, B4129 = CFM analogues; VAN = vancomycin;MET = metronidazole; *Hyper-virulent strain BI/NAP1/027

As shown in Tables 3 and 4, CFM and CFM analogues exhibited excellent invitro activity against all CD strains tested. MICs of CFM and CFManalogues B746, B4157 and B4129 in BHI ranged from ≦0.031 to 1.0 μg/ml,while the MICs of VAN and MET ranged from 0.062 to 2.0 μg/ml (Table 3).The MICs of CFM and analogues B746, B4157 and B4129 in BB ranged from≦0.031 to 0.5 μg/ml, while the MICs of VAN and MET were substantiallyhigher, ranging from 0.125 to 8.0 μg/ml (Table 4).

Antibacterial activity of CFM, analogues B746, B4157 and B4129, VAN andMET against tested CD strains in BB (n=37) is summarized in Table 5below:

TABLE 5 Antibacterial activity of CFM and CFM Analogue activity againstCD CFM B746 B4157 B4129 VAN MET MIC Range (μg/ml) ≦0.031-0.25 ≦0.031-0.5≦0.031-0.25 ≦0.031-0.25 0.25-2.0 0.125-8 MIC₉₀ (μg/ml) 0.25 0.25 0.1250.125 2 4 MIC₅₀ (μg/ml) 0.062 0.125 ≦0.031 ≦0.031 0.5 1 Based on MICdetermined by broth microdilution method in brucella broth (BB); MIC90 =MIC against 90% of strains; MIC50 = MIC against 50% of the strains; n =37;CFM is 8-fold more active than VAN and 16-fold more active than MET.

Antibacterial activity exhibited by CFM against CD was about 8-foldgreater than VAN and about 16-fold greater than MET (Table 5).Similarly, antibacterial activity exhibited by analogues B746, B4157 andB4129 was substantially greater than that of VAN and MET. Based on MIC₉₀values, B746 exhibited 8-fold greater antibacterial activity as comparedto VAN, and 16-fold greater activity as compared to MET. Similarly,B4157 and B4129 exhibited 16-fold greater activity as compared to VAN,and 32-fold greater activity as compared to MET. Thus, CFM and CFManalogues (B746, B4157 and B4129) were determined to be effectiveantimicrobial agents against CD, and exhibited superior antibacterialactivity as compared to both VAN and MET.

Minimum Bactericidal Concentration Screenings:

Minimum bactericidal concentration (MBC) of the test compounds was alsodetermined. 100 μl of the broth from the last three wells or moreshowing no visible growth (FIG. 4, plate A) was spread on brucellaagar/trypticase soy agar plates and incubated in anaerobic chamber at37° C. The colony forming units (CFUs) were counted after 48 hours. Thelowest concentration of the test compound that killed 99.9% of the CDstrain was considered to be the MBC. See Jones et al. (1985)Susceptibility tests: microdilution & macrodilution broth procedures, inBalows, Hausler, Shadomy (eds.) MANUAL OF CLIN. MICROBIOL., Am. Soc'yMicrobiol. Washington, D.C., pp 972-77.

MBC of CFM and VAN was determined against 8 strains of CD, as shown inTable 6:

TABLE 6 MBC of CFM and VAN against C. difficile CFM VAN MIC MBC MBC/ MICMBC MBC/ CD isolate (μg/ml) (μg/ml) MIC (μg/ml) (μg/ml) MIC NR32882 0.250.5 2 1 2 2 NR32883 0.25 0.5 2 1 2 2 NR332884 0.125 0.25 2 1 1 1 NR134300.125 0.25 2 1 1 1 NR13432 0.125 0.25 2 1 2 2 NR13437 0.25 0.25 1 1 1 1VPI10463 0.125 0.25 2 1 4 4 BAA1805* 0.125 0.125 1 0.5 2 4 CFM =clofazimine; VAN = vancomycin; note: vancomycin tolerant colonies weredetected at all concentrations in all the strains;*Hyper-virulent strainBI/NAP1/027

MBC/MIC ratio of CFM ranged from 1-2. MBC/MIC ratio of VAN ranged from1-4. For the hyper-virulent strain, the ratio for CFM is 1 and for VANit is 4.

Combination Screening and Synergistic Activity:

CFM is suitable for use with one or more active compounds, such as incombination therapy, and exhibits a synergistic or additive effect withother active compounds. In particular, CFM in combination with MET, VANand/or FDX exhibits therapeutically effective bactericidal activity, andat a lower dosage as compared to activity of each drug if used alone.Thus, the synergistic effect of such combination enhances activity whilebeneficially reducing dosage.

Inhibitory activity of CFM in combination with either VAN or MET wasdetermined against 5 strains of CD as shown in Table 7:

TABLE 7 In vitro activity of CFM + additional antimicrobial agent(s)against CD MIC (μg/ml) Drug combi- Strain Test: combination alone nationFIC ΣFIC VPI 10463 1 CFM 0.25 0.031 0.124 0.374 VAN 2 0.5 0.25 2 CFM0.125 0.0039 0.0312 0.281 VAN 2 0.5 0.25 3 CFM 0.125 0.062 0.5 1 MET0.125 0.062 0.5 4 CFM 0.125 0.062 0.5 1 MET 0.125 0.062 0.5 NR32889 5CFM 0.125 0.125 1 2 VAN 2 2 1 NR32890 6 CFM 0.125 0.031 0.25 0.75 VAN 10.5 0.5 NR32885 7 CFM 0.125 0.062 0.5 1 VAN 1 0.5 0.5 8 CFM 0.125 0.0310.25 0.375 MET 2 0.25 0.125 9 CFM 0.25 0.062 0.25 0.5 VAN 1 0.25 0.25 10CFM 0.125 0.0039 0.031 0.281 MET 1 0.25 0.25 11 CFM 0.25 0.062 0.25 0.5VAN 1 0.25 0.25 12 CFM 0.25 0.0078 0.031 0.281 MET 1 0.25 0.25 BAA1805*13 CFM 0.25 0.062 0.25 0.312 VAN 0.5 0.031 0.062 14 CFM 0.25 0.00390.015 0.031 MET 16 0.25 0.015 15 CFM 0.25 0.015 0.06 0.185 MET 32 40.125 16 CFM 0.25 0.062 0.25 0.5 VAN 0.5 0.125 0.25 17 CFM 0.25 0.00780.031 0.156 MET 32 4 0.125 NR32888 18 CFM 0.25 0.125 0.5 1 VAN 0.5 0.250.5 19 CFM 0.25 0.015 0.062 0.125 MET 2 0.125 0.062 20 CFM 0.25 0.0620.25 0.75 VAN 0.5 0.25 0.5 21 CFM 0.25 0.0078 0.031 0.28 MET 2 0.5 0.25ΣFIC ≦ 0.5: indicates synergistic or beneficial effect of drugcombination (as compared to effect of drug if used alone); ΣFIC ≧ 4.0:indicates antagonistic or detrimental effect of drug combination (ascompared to effect or activity of each drug if used alone, thusinterfering with effectiveness of each drug alone); ΣFIC > 0.5 but <4.0:indicates additive effect of drug combination (thus indicating drug mayact as substitute for the other).*Hyper-virulent strain BI/NAP1/027

As shown in Table 7, a fractional inhibitory concentration (FIC) isprovided for each drug alone, as well as the total or sum factionalinhibitory concentration (ΣFIC) for each drug combination. As shown bythe data, all drug combinations exhibited either a synergistic effect(in most cases) or an additive effect (in two of the tested cases), ascompared to the activity of each drug alone.

Of note, where clinically virulent strain NAP1/B1/027 MET is becomingineffective, and where MET alone is virtually ineffective against CDstrain BI/NAP1/027 in BB culture (MIC 32 μg/ml), when treated incombination with 0.008 μg of CFM, MIC for MET is reduced to 4 μg/ml forthis virulent CD strain. A similar synergistic effect with FDX islikewise provided against virulent CD strains. Thus, the datademonstrate a substantial reduction in effective dosage requirements ofeach antibiotic if used alone. Moreover, such combination therapysubstantially reduces the potential emergence of resistant CD strains.

CFM and CFM Analogue Activity Against Other Bacterial Species:

In vitro activity of CFM and CFM analogues (B746, B4157 and B4129) wasdetermined against a panel of other bacteria representing a portion ofnormal gut flora, including strains of Escherichia coli (Gram-negative),Enterococcus faecalis, Enterococcus faecium (Gram-positive),Staphylococcus aureus (Gram-positive), Bacteroides spp. (Gram-negativeanaerobic bacteria that are predominant representative normal bacterialflora in the human colon), Fusobacteria spp. (Gram-negative, anaerobic,non-spore forming bacteria), Bifidobacterium spp. (Gram-positive,non-spore forming, anaerobic, branched bacilli), Lactobacilli(Gram-positive, facultative anaerobic, non-spore forming bacteria, alsofound in urogenital canal, in acidic environment) and Lachnospiraceaeand related organisms (Gram-positive, anaerobic bacteria).

As shown in Tables 8-15 below, except for Lachnospiraceae, CFM was noteffective in inhibiting growth of these other tested bacteria atconcentrations demonstrated to be effective against CD. The testedbacterial species, when present in certain proportions in the gut, areknown to resist colonization of CD. See Schubert et al. (2015)Antibiotic-Induced Alterations of the Murine Gut Microbiota & SubsequentEffects on Colonization Resistance against Clostridium difficile,Mbio.asm.org 6(4):e000974-15, pp. 1-10). As such, they are beneficiallymaintained in accordance with the disclosed methods and compositions ofthe present invention.

MICs for CFM, CFM analogues (B746, B4157 and B4129), VAN, MET, CIP, AMPand AMK against Escherichia coli strains are presented in Table 8:

TABLE 8 Antibacterial activity of CFM and CFM Analogues againstEscherichia coli strains MIC (μg/ml) Strain CFM B746 B4157 B4129 VAN METCIP AMP AMK NR 6 >32 >32 >32 >32 >32 >32 ≦0.031 2 4 NR8 >32 >32 >32 >32 >32 >32 ≦0.031 4 4 NR 96 >32 >32 >32 >32 >32 >32≦0.031 4 8 NR 104 >32 >32 >32 >32 >32 >32 ≦0.031 4 2 NR17626 >32 >32 >32 >32 >32 >32 ≦0.031 4 16 NR17627 >32 >32 >32 >32 >32 >32 ≦0.031 4 8 NR17633 >32 >32 >32 >32 >32 >32 ≦0.031 4 8 NR17647 >32 >32 >32 >32 >32 >32 ≦0.031 4 2 NR17650 >32 >32 >32 >32 >32 >32 ≦0.031 4 8 NR17661 >32 >32 >32 >32 >32 >32 >32 >32 2 MIC determined by microdilutionmethod in Mueller-Hinton broth; CFM = clofazimine; B746, B4157, B4129 =CFM analogues; VAN = vancomycin; MET = metronidazole; CIP =ciprofloxacin (Fluoroquinolones); AMP = ampicillin (Beta-Lactam); AMK =amikacin (aminoglycoside)

MICs for CFM, CFM Analogues (B746, B4157 and B4129), VAN, MET, CIP, AMPand AMK against Enterococci faecalis and Enterococci faecium strains arepresented in Table 9. As shown, the activity demonstrated by CFM andB746 against Enterococci faecium is far superior to that demonstrated byconventional drugs tested (e.g., MET, VAN and FDX). Thus, CFM and CFManalogues (and in particular B746) are suitable and advantageouscandidates for effectively treating CDAD, while also minimizing the riskof emergence of resistant strains (e.g., VRE). Indeed, despite itslong-term use in the treatment of leprosy and Mycobacterium tuberculosis(MTB), CFM has shown negligible incidences of emergence of resistantstrains.

TABLE 9 Antibacterial activity of CFM and CFM Analogue activity againstEnterococcus faecalis and Enterococci faecium strains Species & MIC(μg/ml) Strain CFM B746 B4157 B4129 VAN MET CIP AMP AMK E. faecalis 324 >32 >32 2 >32 1 4 >32 NR 31884 E. faecalis 32 4 >32 >32 2 >32 0.54 >32 NR 31885 E. faecalis 32 8 >32 >32 1 >32 0.5 4 >32 NR 31886 E.faecalis 16 4 >32 >32 1 >32 0.5 0.5 >32 NR 31887 E. faecalis 328 >32 >32 2 >32 1 4 >32 NR 31888 E. faecium 42 >32 >32 >32 >32 >32 >32 >32 NR 31903 E. faecium 22 >32 >32 >32 >32 >32 >32 >32 NR 31909 E. faecium 22 >32 >32 >32 >32 >32 >32 >32 NR 31912 E. faecium 16 2 >32 >32 >32 >32 42 >32 NR 31915 E. faecium 8 2 >32 >32 >32 >32 1 >32 >32 NR 31916 MICdetermined by microdilution method in Mueller-Hinton broth; CFM =clofazimine; B746, B4157, B4129 = CFM analogues; VAN = vancomycin; MET =metronidazole; CIP = ciprofloxacin; AMP = ampicillin; AMK = amikacin.

MICs for CFM, analogues B746, B4157 and B4129, VAN, MET, CIP, AMP andAMK against Staphylococcus aureus strains are presented in Table 10:

TABLE 10 Antibacterial activity of CFM and CFM Analogues againstStaphylococcus aureus strains MIC (μg/ml) Strain: CFM B746 B4157 B4129VAN MET CIP AMP AMK NR 10129 16 4 32 32 1 >32 2 >32 2 NR 10186 2 216 >32 1 >32 32 >32 32 NR 10187 >32 4 >32 >32 2 >32 >32 8 >32 NR 10188 84 32 >32 1 >32 >32 16 >32 NR 10189 8 4 >32 >32 2 >32 1 32 4 NR 10191 4 432 >32 1 >32 >32 >32 32 NR 10192 8 4 >32 >32 1 >32 >32 32 >32 NR 10193 44 32 >32 2 >32 >32 >32 >32 NR 10194 4 4 16 32 1 >32 0.5 2 32 NR 10198 1616 >32 >32 2 >32 >32 >32 >32 MIC determined by microdilution method inMueller-Hinton broth; CFM = clofazimine; B746, B4157, B4129 = CFManalogues; VAN = vancomycin; MET = metronidazole; CIP = ciprofloxacin;AMP = ampicillin; AMK = amikacin

MICs for CFM, analogues B746, B4157 and B4129, AZQ, VAN and MET againstBacteroides strains are presented in Table 11:

TABLE 11 Antibacterial activity of CFM and CFM Analogues againstBacteroides Strains MIC (μg/ml) Species: Strain: CFM B746 B4157 B4129AZQ VAN MET B. spp. HM18 4 1 2 16 2 >32 4 B. spp. HM19 1 2 0.5 0.5 4 >321 B. spp. HM20 2 1 2 2 2 16 0.25 B. spp. HM22 2 1 1 2 2 32 0.5 B. spp.HM23 2 2 2 16 2 8 0.25 B. spp. HM27 1 1 0.5 1 1 16 0.5 B. spp. HM28 2 22 2 2 >32 2 B. spp. HM58 1 1 1 2 4 32 2 B. eggerthii HM210 2 2 1 2 2 320.5 B. ovatus HM222 0.25 0.25 0.25 0.25 1 4 0.5 B. fragilis HM709 4 4 22 2 8 1 B. fragilis HM710 2 2 2 2 2 8 0.5 B. fragilis HM714 2 4 2 4 2 161 B. caccae HM728 1 1 2 2 2 32 0.25 B. stercoris HM1036 0.5 1 0.5 0.25 216 0.25 MIC determined by microdilution method in Brucella broth; CFM =clofazimine; B746, B4157, B4129 = CFM analogues; AZQ = azaquinone; VAN =vancomycin; MET = metronidazole

As shown by data presented in Tables 8, 9 and 10 above, CFM and CFManalogues B746, B4157 and B4129 were relatively inactive against E.coli, and relatively inactive to only modestly active against otherGram-positive bacteria with higher MICs. All tested compounds were lessactive than VAN against Enterococcus and S. aureus strains tested. Asshown in Table 11, CFM and CFM analogues also exhibited good in vitroactivity against Bacteroides strains (e.g., wherein antibacterialactivity was significantly superior to VAN and comparable to MET).

As shown above, CFM kills Bacteroides spp. in in-vitro assays. Crohn'sdisease patients have a preponderance of these Gram-negative anaerobicbacteria in their guts as compared to normal population. See Bibiloni etal. (2006) The bacteriology of biopsies differs between newly diagnosed,untreated, Crohn's disease and ulcerative colitis patients, J. Med.Microbiol. 55:1141-49. As demonstrated herein, CFM's significant effecton the disease is by inhibiting the growth of these Bacteroides in thegut of patients with Crohn's disease, when used alone (see Afdhal et al.(1991) Controlled trial of anti-microbial therapy in Crohn's disease:clofazimine versus placebo, Dig. Dis. Sci. 36:449-53) or when used incombination therapies (Prantera et al. (1994) Antimycobacterial therapyin Crohn's Disease: results of a controlled double blind trial with amultiple antibiotic regimen, Am. J. Gastroentrol. 89:513-18; Selby etal. (2007) Two year combination antibiotic therapy with clarithromycin,rifabutin, and clofazimine for Crohn's disease, Gastroenterol.132:2313-19.

CFM and CFM analogues show limited or no in vitro activity againstFusobacterium spp., as shown in Table 12 below. Fusobacteria areGram-negative, anaerobic, non-spore forming bacteria that are part ofthe normal flora of the gastrointestinal tract. Moderate activity isseen for HM-993 strain, though such activity is at least 8-fold lessthan that observed against a majority of CD strains. VAN was completelyinactive against these strains (MIC>32 μg/ml). However, MET displayedexcellent activity with MIC ranging from ≦0.031-0.5 μg/ml.

TABLE 12 Antibacterial activity of CFM and CFM Analogues againstFusobacterium MIC (μg/ml) Species: Strain # CFM B746 B4157 B4129 VAN METCIP Fusobacterium sp. HM 42 >32 >32 >32 >32 >32 0.125 — Fusobacteriumsp. HM 57 >32 >32 >32 >32 >32 0.5 — Fusobacterium sp. HM-556 168 >32 >32 >32 0.125 2 Fusobacterium sp. HM-758 >32 >32 >32 >32 >32 0.1251 Fusobacterium sp. HM-871 32 8 >32 >32 >32 0.125 2 Fusobacterium sp.HM-874 >32 16 >32 >32 >32 0.125 2 Fusobacterium sp. HM-875 328 >32 >32 >32 0.125 1 F. nucleatum HM-75 >32 16 >32 >32 >32 0.062 1 F.nucleatum HM-260 >32 >32 >32 >32 >32 0.125 1 F. nucleatum HM-992 >3216 >32 >32 >32 0.125 1 F. nucleatum HM-993 2 2 4 8 >32 ≦0.031 1 F.nucleatum HM-994 32 8 >32 >32 >32 0.125 1 F. nucleatum HM-995 >3216 >32 >32 >32 0.125 2 F. nucleatum HM-996 32 32 >32 >32 >32 0.5 2 F.nucleatum HM-997 >32 16 >32 >32 >32 0.25 1 MIC determined bymicrodilution method in Brucella broth; CFM = clofazimine; B746, B4157,B4129 are CFM analogues; VAN = vancomycin; MET = metronidazole; CIP =ciprofloxacin

CFM showed modest or no activity against Bifidobacterium spp., as shownin Table 13 below. Bifidobacterium spp. are Gram-positive, non-sporeforming, anaerobic, branched bacilli that are also part of the normalflora of the gastrointestinal tract. CFM analogues showed modest invitro activity against nine of the ten strains tested (MIC 0.5-2 μg/ml)and no activity against one strain tested. Tested CFM analogues werefound to be less active than CFM (Table 13). Except for one resistantstrain, VAN displayed high activity against these bacteria, with MICranging from 0.25 to 1 μg/ml. MET activity was variable, with four ofthe ten strains susceptible.

Overall, the data show that strains of Bifidobacterium longum speciestested are more susceptible to this group of antibiotics than otherspecies, suggesting the utilization of individualized patienttreatments. For example, a patient harboring strain HM-845 ofBifidobacterium longum may be best served by using CFM analog B4157compared to the use of other antibiotics for CD treatment.

TABLE 13 Antibacterial activity of CFM and CFM Analogues againstBifidobacterium strains MIC (μg/ml) Species Strain: CFM B746 B4157 B4129VAN MET CIP Bifidobacterium sp. HM-30 0.5 1 8 4 0.5 >32 >32Bifidobacterium breve HM-411 2 4 16 >32 0.5 >32 4 Bifidobacterium breveHM-412 2 4 4 0.25 0.5 >32 4 Bifidobacterium adolescentis HM-633 1 216 >32 0.5 >32 8 Bifidobacterium longum HM-845 1 2 32 ≦0.031 0.5 2 4Bifidobacterium longum HM-846 2 2 8 0.25 0.5 2 4 Bifidobacterium longumHM-847 1 2 1 2 0.5 1 8 Bifidobacterium breve HM-856 4 32 >32 >321 >32 >32 Bifidobacterium sp. HM-868 2 2 32 >32 0.25 16 0.5Bifidobacterium breve HM-1120 >32 >32 >32 >32 8 0.5 >32 MIC determinedby microdilution method in BHI broth; CFM = clofazimine; B746, B4157,B4129 are CFM analogues; VAN = vancomycin; MET = metronidazole; CIP =ciprofloxacin

In vitro activity of CFM and its analogues against Lactobacillus spp.was variable, as shown in Table 14. Lactobacilli are Gram-positive,facultative anaerobic, non-spore forming bacteria normally live ingastrointestinal and urogenital tracts, in acidic environment,preventing colonization of pathogenic organisms. Of the ten speciesstrains tested, six were resistant to the CFM analogues, and four weremoderately susceptible. However, MIC values against these 4 strains werestill 4 to 8 fold higher than those required to inhibit CD by CFM.Similarly, in vitro activity of VAN was variable, with six strainssusceptible and four resistant. MET was essentially inactive against allstrains tested.

TABLE 14 Antibacterial activity of CFM and CFM Analogues againstLactobacillus strains MIC (μg/ml) Species: Strain: CFM B746 B4157 B4129VAN MET CIP Lactobacillus rhamnosus HM-106 >32 >32 >32 >32 >32 >32 2Lactobacillus sp. HM-228 1 4 ≦0.031 1 1 >32 4 Lactobacillus gasseriHM-104 >32 16 >32 >32 1 >32 >32 Lactobacillus jenseniiHM-105 >32 >32 >32 >32 1 >32 32 Lactobacillus crispatus HM-637 4 8 2 160.5 >32 >32 Lactobacillus iners HM-126 2 4 2 1 1 >32 2 Lactobacillusvaginalis HM-405 2 4 2 2 >32 >32 >32 Lactobacillus sp. HM-478 >3216 >32 >32 >32 >32 >32 Lactobacillus jhonsonii HM-643 >32 32 >32 >322 >32 >32 Lactobacillus reuteri HM-102 >32 32 >32 >32 >32 >32 >32 MICdetermined by broth microdilution method in BHI with 0.02% Tween 80, pH6.0; CFM = clofazimine; B746, B4157 & B4129 are CFM analogs; VAN =vancomycin; MET = metronidazole; CIP = ciprofloxacin.

In vitro activity of CFM and its analogues against Lachnospiraceae spp.is presented in Table 15. Lachnospiraceae and related organisms areGram-positive, anaerobic bacteria that are part of normal flora of thegut. Most of the members of this family were found to be susceptible toCFM, CFM analogues, VAN and MET. Their susceptibility of ciprofloxacinwas variable.

TABLE 15 Antibacterial activity of CFM and CFM Analogues againstLachnospiraceae and related bacterial strains MIC (μg/ml) Species Strain# CFM B746 B4157 B4129 VAN MET CIP Lachnospiraceae sp. HM-150 0.25 0.50.25 0.125 1 0.25 16 Lachnospiraceae sp. HM-153 0.125 0.5 0.25 0.125 40.062 4 Lachnospiraceae sp. HM-480 0.062 0.125 ≦0.031 0.062 0.25 0.5 16Lachnospiraceae sp. HM-157 0.125 0.5 0.5 0.25 0.5 0.25 >32Lachnospiraceae sp. HM-161 0.25 0.25 0.125 0.125 0.5 0.125 32Lachnospiraceae sp. HM-7 ≦0.031 ≦0.031 ≦0.031 ≦0.031 0.125 0.25 8Lachnospiraceae sp. HM-154 0.5 0.5 0.5 0.125 0.5 0.125 16Lachnospiraceae sp. HM-558 0.062 0.125 0.062 0.125 0.25 0.5 16Lachnospiraceae sp. HM-768 ≦0.031 ≦0.031 ≦0.031 ≦0.031 0.125 0.5 8Lachnospiraceae sp. HM-868 2 2 32 >32 0.25 16 0.5 Clostridialesbacterium HM-182 0.25 1 0.5 0.125 0.25 ≦0.031 8 Eubacterium sp. HM-1780.125 0.5 1 0.125 2 1 0.5 Peptostreptococcaceae sp. HM-766 1 1 0.5 0.50.5 0.5 0.25 Clostridiales bacterium HM-84 ≦0.031 0.062 ≦0.031 ≦0.0310.5 0.25 16 MIC determined by broth microdilution method in brucellabroth; CFM = clofazimine; B746, B4157 and B4129 are CFM analogs; VAN =vancomycin; MET = metronidazole; CIP = ciprofloxacin.AZQ Activity Against CD Strains:

AZQ exhibited excellent antibacterial activity against CD strains, withvery narrow MIC range (0.25-1 μg/ml). AZQ activity was comparable to VANand superior to MET. MIC data for AZQ, VAN and MET are presented inTable 16:

TABLE 16 MIC of AZQ against CD strains MIC (μg/ml) Strain: AZQ VAN METVPI 10463 0.5 1 0.125 HM88 1 0.5 1 HM89 1 0.5 2 HM745 1 1 2 HM746 1 1 2HM747 1 0.5 0.5 NR13427 1 2 8 NR13428 1 1 0.5 NR13429 1 0.5 4 NR134300.5 1 1 NR13431 1 0.5 2 NR13432 1 2 2 NR13433 1 0.5 0.125 NR13434 0.250.5 0.5 NR13435 1 0.5 0.5 NR13436 1 2 2 NR13437 1 0.5 1 NR13438 1 2 2NR13553 1 0.5 0.5 NR32882 1 0.5 0.5 NR32883 0.5 0.5 1 NR32884 1 1 0.5NR32885 1 2 1 NR32886 1 1 1 NR32887 1 1 8 NR32888 1 0.5 1 NR32889 1 2 8NR32890 0.5 1 1 NR32891 1 0.5 1 NR32892 1 0.5 2 NR32895 1 2 4 NR32896 12 1 NR32897 1 0.25 2 NR32900 1 0.5 1 NR32903 1 0.5 0.5 NR32904 1 0.5 2MIC determined by broth microdilution method in brucella broth; AZQ =azaquinone; VAN = vancomycin; MET = metronidazole; MIC₉₀ of AZQ = 1μg/ml, VAN = 2 μg/ml and MET = 4 μg/ml.AZQ Activity Against Other Bacterial Strains:

Antibacterial activity of AZQ was also tested against other bacteriarepresenting normal gut floral. AZQ exhibited no activity againstGram-negative bacteria (E. coli) and relatively low levels of activityagainst other Gram-positive bacteria. MIC data for AZQ, VAN, CIP, AMPand AMK against E. coli, Staphylococcus aureus, Enterococcus faecalis,and Enterococcus faecium are presented in Table 17. AZQ exhibited goodin vitro activity against Bacteroides spp. (see Table 11), whereinantibacterial activity was significantly superior to VAN and comparableto MET).

TABLE 17 Activity against other facultative anaerobic bacteria MIC(μg/ml) Organism Strain AZQ VAN CIP AMP AMK E. coli NR 6 >32 >32 ≦0.0312 4 NR 8 >32 >32 ≦0.031 4 4 NR 96 >32 >32 ≦0.031 4 8 NR 104 >32 >32≦0.031 4 2 NR 17626 >32 >32 ≦0.031 4 16 NR 17627 >32 >32 ≦0.031 4 8 NR17633 >32 >32 ≦0.031 4 8 NR 17647 >32 >32 ≦0.031 4 2 NR 17650 >32 >32≦0.031 4 8 NR 17661 >32 >32 >32 >32 2 S. aureus NR 10129 32 1 2 >32 2 NR10186 16 1 32 >32 32 NR 10187 >32 2 >32 8 >32 NR 10188 32 1 >32 16 >32NR 10189 16 2 1 32 4 NR 10191 8 1 >32 >32 32 NR 10192 32 1 >32 32 >32 NR10193 16 2 >32 >32 >32 NR 10194 16 1 0.5 2 32 NR 10198 16 2 >32 >32 >32E. faecalis NR 31884 32 2 1 4 >32 NR 31885 32 2 0.5 4 >32 NR 31886 32 10.5 4 >32 NR 31887 16 1 0.5 0.5 >32 NR 31888 32 2 1 4 >32 E. faecium NR31903 >32 >32 >32 >32 >32 NR 31909 32 >32 >32 >32 >32 NR 3191232 >32 >32 >32 >32 NR 31915 >32 >32 4 2 >32 NR 31916 16 >32 1 >32 >32MIC determined by broth microdilution method in Mueller-Hinton broth.AZQ = azaquinone; VAN = vancomycin; CIP = ciprofloxacin; AMP =ampicillin; AMK = amikacin.Animal Model Testing and Observations:

For the treatment of CDAD, CFM is retained in the gut in atherapeutically sufficient concentration and sufficient period of timefor clearing CDI. Animal model tests (e.g., Syrian Golden Hamster model)were conducted utilizing three doses of CFM in aqueous suspension ofcarboxy methyl cellulose (CMC), administered orally to the animals (10,20 and 40 mg/kg body weight/day, once a day) for 5 days.

Efficacy of CMC formulation with Lamprene® at doses 10 mg/kg and 20mg/kg twice a day were compared. Efficacy of a combination of Lamprene®at 10 mg/kg+VAN at 5 mg/kg (administered orally twice a day) was alsocompared with VAN at 5 mg/kg alone and with Lamprene® at 10 mg/kg alone(administered orally twice a day). Data from these experiments conformedto results demonstrated by in vitro testing.

At lower doses of CFM of 10 mg/kg/day, 50% protection was observed onanimal survival by day 9 after infection was achieved. In contrast, allten animals in the control groups died by day 10. Beyond 10 days,protection diminished for some animals. 30% protection was observed forCFM dosage of 10 mg/kg during 28 days of observation. However, higherdosages of 20 and 40 mg/kg did not increase the protection.

As expected, treatment of animals with CFM in aqueous suspension of CMCat 10 mg/kg/once a day was demonstrated to be more promising (30%survival for 27 days) compared to survival of animals treated withLamprene®, which is intended for systemic absorption, at 10 mg/kg or 20mg/kg twice a day (0% survival by 7 days). In addition, animal groupstreated with a combination of Lamprene® (lower dose) with a low dose ofVAN fared much better (40% survival) compared to groups treated witheach drug alone at the same dosage (0% survival by day 7 for CFM and 0%survival by day 14 for VAN).

Thus, dosage used for some experiments was too high. According to dosetranslation calculations from humans to animals, 20 mg/kg for hamstersis the upper tolerable limit (see Reagan-Shaw et al. (2007) Dosetranslation from animal to human studies revisited, FASEB J. 22,659-61), though such limit may be too high for animals challenged withCD. Thus, a dosage of 5-7 mg/kg is more suitable.

CFM Solubility:

CFM is poorly soluble in water. Thus, CFM is typically administered inan oil-based formulation, wherein the drug is suspended in a mixture ofoils and emulsifying agents in a gel-capsule. In the treatment ofleprosy and multi-drug resistant TB (MDR-TB), such formulation is alsotypically optimized for better systemic absorption.

However, the success treatment of CD requires that the drug stay in thegut for a much longer period as compared to treatments for leprosy andMDR-TB. Despite excellent in vitro activity against CD, relatively lowtherapeutic efficacy in hamster models was seen in preliminary studiesdue to poor CFM solubility and availability in the presence of fecalmatter. Several formulations were therefore developed for effective invivo dosing for CD therapies (Table 18), which exhibit improvedsolubility and efficacy in the gut in the presence of fecal matter.

TABLE 18 CFM formulations suitable for in vivo dosing Form and Agentstrength Solubility Stability Comments Polyethylene Liquid, 5 mg/mlStable undiluted, the Works well in in vitro glycol 100% drug opalescentstudies; however, because (PEG-300, following dilution in the drugprecipitates upon PEG-400) water or medium; dilution, it may not beuseful precipitates faster in for in vivo studies. saline/PBSCremophor-EL Liquid, 8 mg/ml Stable, and remains Most stable formulationand 100% 10 mg/ml if stable following remains clear upon dilution keptfor long dilution in water/ and most suitable for in vitro time (3-5medium/saline/PBS and in vivo studies, can also days) used for in vitrostudies. Tween 80 Liquid, 12 mg/ml, Stable undiluted, Maximum solubility100% 15 mg/ml if stable after dilution in compared to any other kept forlong water or culture solvent tested. Stable and time medium, PBS,remains clear upon dilution (3-5 days) precipitates slowly and suitablefor both in vitro in saline. and in vivo studies. Phosal 53 MCT Thickoily 10 mg/ml Stable, clear, thick Useful only for in vivo(phospholipids) liquid solution. Should be drug dosing diluted in oilsor phospholipids. Forms emulsion in water Phosal 50 PG Thick oily 10mg/ml Stable, clear, thick Useful only for in vivo (phospholipids)liquid, solution. drug dosing. slightly Should be diluted in thinnerthan oils or phospholipids. Phosal 53 Forms emulsion MCT in water Oliveoil, Liquid, 5 mg/ml Stable, should be Not useful for in vitro canolaoil, and 100% diluted only in oils, studies; useful for in vivo coconutoil forms emulsions drug dosing in water

In vitro activity of CFM formulations according to the present inventionwas determined against CD, with all formulations exhibiting excellentactivity (Table 19). MICs were similar to DMSO solubilized drug used inour in vitro studies.

TABLE 19 In vitro activity of CFM-formulations Drug/formulation MIC(μg/ml) MBC (μg/ml) CFM-DMSO 0.25 0.25 CFM-PEG 0.125 0.125 CFM-CrEL0.125 0.125 CFM-Tween 0.125 0.125 VAN 0.5 2.0 MET 0.25 0.25 Organism andstrain: CD strain VPI 10463; Medium used: BHI; CFM = clofazimine; DMSO =dimethyl sulfoxide; PEG = polyethylene glycol; CrEL = cremophor EL;Tween = Tween 80; VAN = vancomycin; MET = metronidazole.

Drugs may bind to organic matter in the body and lose their activity. Tobe effective, the compound should display antibacterial activity againstCD in the presence of fecal matter. To determine the effect of solublefecal matter on in vitro activity of CFM, normal healthy human feces wassuspended in BHI broth at 20% w/v, mixed well and filter sterilized. MICof CFM was determined in culture medium containing 20% fecal extracts.No significant deviation in MIC was found. A one to two fold increase inMIC was demonstrated from experimental manipulations, as shown in Table20 below:

TABLE 20 Effect of fecal extracts on the MIC of CFM against C. difficileVPI10463 MIC (μg/ml) Expt. 1 Expt. 2 Drugs: −fecal extract fecal extract−fecal extract +fecal extract CFM 0.125 0.5 0.062 0.125 VAN 0.5 1 0.50.5 MET 0.062 0.125 0.062 0.125 Human feces (20% w/v) was extracted inBHI broth, filter sterilized and used for testing MIC. Drug dilutionsprepared in the fecal extracts were incubated for 4 hrs in anaerobicincubator before adding bacterial suspension.

Subsequently, different CFM formulations (CFM-DMSO, CFM-F68, CFM-PEG andCFM-CrEL) were added to 2 ml volumes of 20% feces in BHI broth to give128 μg/ml concentrations and incubated for 2 hours at 37° C. Uponcentrifugation at 3000 rpm for 15 minutes, insoluble fecal matterpelleted at the bottom, leaving supernatant liquid phase on top. Thesupernatants of CFM-DMSO, CFM-F68 and CFM-PEG formulations wereyellowish in color (FIG. 9 and FIG. 10), similar to BHI broth color,indicating most of the drug in these sample tubes pelleted along withparticulate fecal matter leaving little or no drug in the liquid phase.In contrast, the supernatant of feces incubated with CFM-CrEL was deeporange in color (FIG. 9 and FIG. 10), suggesting the presence of drug inthe liquid phase. Afterward, the supernatants were transferred intomicrocentrifuge tubes, spun at 5000 rpm for 10 minutes and filtersterilized through 0.45 μm followed by 0.22 μm filters. Even though somedrug bound to the filter membrane, the supernatant of feces incubatedwith CFM-CrEL remained deep orange (FIG. 10), indicating the presence ofsoluble CFM.

The supernatants of feces incubated with the other three formulationsremained yellowish, similar to the color of BHI broth. The MIC of thefiltered supernatants was determined by a broth microdilution methodagainst CD. CFM formulations prepared in BHI broth, withoutcentrifugation and after centrifugation, served as controls (Table 21).

CFM formulations in DMSO, PEG and CrEL showed similar activity in BHIcontrol with MIC of 0.0625 μg/ml (Table 21, column 1). Centrifugation ofthe CFM formulations prepared in BHI resulted in sedimentation ofinsoluble drug and reduction of MIC. The extent of reduction was highestfor CFM-F68, followed by CFM-DMSO, CFM-PEG and CFM-CrEL, respectively(Table 21, column 2). Incubation of CFM-DMSO, CFM-F68 and CFM-PEGformulations with 20% feces resulted in binding of CFM to particulatematter in the feces with little or no drug in the liquid phase uponcentrifugal separation and displayed no antibacterial activity, with MICreaching >32 μg/ml. CFM-CrEL formulation, on the other hand, showedhighest activity with MIC of 0.25 μg/ml (Table 21, column 3). Incubationin feces did not substantially affect the MIC of VAN, which exhibitedonly a 2-fold increase. However, MIC of MET increased significantly from0.125 to >32 μg/ml. suggesting that MET also binds to particulate fecalmatter, thereby causing a significant reduction in antibacterialactivity.

TABLE 21 In vitro activity of CFM formulations following incubation withfeces MIC (μg/ml) in Centrifuged Drug/formulation BHI control* BHIcontrol** Fecal extracts*** CFM-DMSO 0.062 1 >32 CFM-F68 0.25 32 >32CFM-PEG 0.062 0.5 >32 CFM-CrEL 0.062 0.25 0.25 VAN 0.5 0.5 1.0 MET 0.0620.125 >32 MIC was determined by broth microdilution method in BHI brothagainst CD strain VPI 1063. CFM = clofazimine; DMSO = dimethylsulfoxide; F68 = Pluronic diblock polymer; PEG = polyethylene glycol400;CrEL = cremophor EL; VAN = vancomycin; MET = metronidazole. *Serial drugdilutions were prepared in BHI tested for activity withoutcentrifugation. **Drugs were diluted in BHI to give 128 μg/mlconcentration, incubated for 2 hours at 37° C., centrifuged at 5000 rpmfor 10 minutes, supernatants were diluted serially and tested foractivity. ***Drugs were diluted in 20% feces to give 128 μg/mlconcentration, incubated for 2 hours at 37° C., centrifuged at 5000 rpmfor 10 minutes, supernatants were filter sterilized, diluted seriallyand tested for activity.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

We claim:
 1. A method for inhibiting the activity of Clostridiumdifficile in a subject, comprising administering to said subject acombination of clofazimine and metronidazole, wherein said combinationexhibits a synergistic effect of increased inhibition of the activity ofClostridium difficile at a lower dose as compared with eitherclofazimine or metronidazole administered alone.
 2. The method of claim1, wherein the subject suffers from a Clostridium difficile associateddisease or a Clostridium difficile infection selected from the groupconsisting of diarrhea, ulcerative colitis and Crohn's disease.
 3. Themethod of claim 1, wherein said clofazimine is administered in apharmaceutical dosage form comprising from 0.0039 μg/ml to 0.031 μg/mland said metronidazole is administered in a pharmaceutical dosage formcomprising from 0.125 μg/ml to 4.0 μg/ml.
 4. The method of claim 3,wherein said clofazimine is administered in a pharmaceutical dosage formcomprising 0.008 μg/ml and said metronidazole is administered in apharmaceutical dosage form comprising 4.0 μg/ml.
 5. The method of claim1, wherein said combination is administered orally.
 6. The method ofclaim 1, wherein said combination is administered simultaneously.
 7. Themethod of claim 1, wherein said combination is administeredsequentially.
 8. The method of claim 1, wherein said combination isadministered in the same pharmaceutical formulation.
 9. The method ofclaim 1, wherein said method further comprises administering at leastone additional therapeutic agent.
 10. The method of claim 9, whereinsaid additional therapeutic agent is an antimicrobial agent.
 11. Themethod of claim 10, wherein said antimicrobial agent is vancomycin orfidaxomicin.
 12. A method for inhibiting the activity of Clostridiumdifficile in a subject, comprising administering to said subject apharmaceutical dosage form consisting of a combination of clofazimineand metronidazole and at least one pharmaceutically acceptableexcipient, wherein said pharmaceutical dosage form comprises from 0.0039μg/ml to 0.031 μg/ml clofazimine and from 0.125 μg/ml to 4.0 μg/mlmetronidazole, and wherein said combination exhibits a synergisticeffect of increased inhibition of the activity of Clostridium difficileat a lower dose as compared with either clofazimine or metronidazoleadministered alone.
 13. The method of claim 12, wherein the subjectsuffers from a Clostridium difficile associated disease or a Clostridiumdifficile infection selected from the group consisting of diarrhea,ulcerative colitis and Crohn's disease.
 14. The method of claim 12,wherein said method further comprises administering at least oneadditional therapeutic agent.
 15. The method of claim 14, wherein saidadditional therapeutic agent is an antimicrobial agent.
 16. The methodof claim 15, wherein said antimicrobial agent is vancomycin orfidaxomicin.